Lipoprotein TFP
1.3 The factor VII protein, gene and cDNA
1.3.1 Structure and function
Factor VII plays a key role in coagulation and its activation is the primary event in blood coagulation (Figure 1.1 & 1.2). Factor VII is synthesised in the liver and circulates in plasma as a single chain 50 kDa glycoprotein of 406 amino acids (Figure 1.5). It is found in the plasma at a concentration of 0.5 pg/ml, approximately 10 nM, with the shortest half-life o f the classical coagulation factors at 3-4 hrs (Fair, 1983).
Ihtron G
'vM >«
H L factor Xa D N s’' w" J Intron E (131) HC^C? t TP TV EY Q Ihtron D (84) C - CkP 63 Intron C (46) J Intron B -38 M T H Y R G T W y (37/38) GD S VwA I ^ M P406 At V G H F COOH ^ ® Intron A (-17) ^ ^ YrYl S G P R L Y Y L F a n( ^ ^r r r h l v g h a e e q t v f v a a l c®DOMAIN
Protease
PRE-PROLEADER
Figure 1.5: Amino acid sequence of pre-pro-factor VII.
Residues ringed are as follows: R 152,1153, activation scissile bond; H193, D242, S344, the catalytic triad. The light arrows denote nonsense and missense mutations associated with factor VII deficiency. The heavy arrows denote the location of the introns. y, y- carboxyglutamic acid residues; p, p-hydroxyaspartic acid. Reproduced from Cooper et a l (1997).
The complete amino acid sequence of fVII (Figure 1.5) was inferred from a full- length cDNA clone isolated by Hagen et a l, in 1986. It is synthesised with a 38 amino acid prepro leader sequence which contains a hydrophobic signal domain (residues -36 to -24) targeting the protein for secretion and a prosequence (residues -1 to -17 ) signalling for the y-carboxylation of ten residues (6, 7, 14, 16, 19, 20, 25, 26, 29 & 35) thereby forming the Gla domain of the protein. The pre-pro sequence is highly conserved in the six human vitamin K-dependent proteins: prothrombin, factors VII, IX and X and proteins C and S (Figure 1.6). The mature protein is produced by cleavage of an Arg-Ala bond; this serves to remove the propeptide prior to secretion into the circulation and leaves a single chain of 406 residues. The post-translational y- carboxylation o f the vitamin K-dependent proteins during protein synthesis is shown in Figure 1.7.
Figure 1.6: Pre-pro sequence of six human vitamin K-dependent proteins.
30 20 10
U)
K)
Pro thro m bin
F a c to r VII F a c to r IX F a c to r X P ro tein C P ro tein S V 1 R M G L Q L P G C L A L A A L C S L V H M A G C A V S Q A L R L L C L L L G L Q E S P G L 1 T 1 C L L G Y L L S M G R P L H L V L L S A S L A G L L L L Q L T S L L L F V A T W G 1 S G T P A P R V L G G R C G A P L A C L L L V L P V Q H V F A A V F C T V F E S L F D S V F E A N L V T Q Q Q A R S L L Q R V R R E E A H G B L H R R R R E N A N K 1 L N R P K R E Q A N B 1 L A R V T R E R A H Q V L R 1 R K R Q Q A S Q V L 0 R R -1 1 A
The vertical boxes show conserved residues thought to be essential for recognition by microsomal y-carboxylase ensuring proper y- carboxylation; the horizontal boxes show the hydrophobic core of the pre-sequence essential for secretion into the endoplasmic reticulum. Reproduced from Tuddenham & Cooper. (1994).
Growing p o p tld o chain
Signal racognltlon part I d a—
Maaaangar RNA fla c a p to r:
algnal raco g n ltlo n partlcla /rlb o a o m a e o m p la x--- ENDOPLASMIC RETICULUM Rropaptlda CYTOPLASM 'f-C arboxylatlon Trans golgl PLASMA
Figure 1.7: Post-translational y-carboxylation of the vitamin K-dependent proteins during protein synthesis. The signal recognition particle binds to the signal peptide, leading to the formation of a ribosome-particle-messenger RNA complex on the endoplasmic reticulum. The signal peptide is translocated to the luminal aspect o f the rough endoplasmic reticulum. After signal peptide cleavage, the propeptide is expressed on the nascent polypeptide chain. The propeptide, containing the y-carboxylation recognition site binds to the vitamin K-dependent carboxylase associated within the endoplasmic reticulum. Specific glutamic acids are converted to y-carboxyglutamic acids, then the protein is transported to the trans-golgi where the propeptide is cleaved. The fully processed protein is secreted into the circulation. Reproduced from Furie & Furie. (1990).
Unlike the other serine proteases o f the coagulation cascade (thrombin, factor IX, factor X & factor XII), the fVII protein has no, or virtually no, activity in the absence o f tissue factor (TF). Factor VII becomes activated to factor V ila by factor Xa (Butenas & Mann, 1996, Radcliffe & Nemerson, 1975 & 1976). It has been reported that the serine proteases, factor Xlla (Broze & Majerus, 1980, Kisiel et al, 1977, Radcliffe et al, 1977 and Seligsohn et al, 1979), factor IXa (Seligsohn et a l, 1979) and thrombin (Radcliffe & Nemerson, 1975) are also biologically relevant for factor VII activation, although this is still controversial. Activation results in the cleavage o f a single internal bond between arginine and isoleucine at residues 152/153, a step which does not require tissue factor (Butenas & Mann, 1996). The cleavage leads to the formation of an enzyme composed of two polypeptide chains; a light chain o f 152 amino acids and a heavy chain of 254 amino acids held together by a disulphide bond from Cysl35 to Cys262. The light chain contains the y-carboxyglutamic acid (Gla) domain, an amphipathic helix and two epidermal growth factor like domains, while the heavy chain contains the catalytic triad His 193, Asp242 and Ser344, typical o f the serine proteinases.
When activated, the tissue factor:factor Vila complex is then capable of activating factors IX and X and autocatalytically factor VII itself. Activated factor VII still requires calcium ions and tissue factor to exhibit significant proteolytic activity. It was a long held belief that factor X was the primary physiological substrate of the factor Vlla/tissue factor complex. However, detailed re-evaluation of the kinetics of activation of factor IX versus factor X by factor Vlla/tissue factor suggests that factor IX is the more important substrate (Komiyama et al, 1990, Repke et al, 1990). This explains the reduced level of factor Vila associated with haemophilia B (Wildgoose et a l, 1992) and
the serious bleeding disorder associated with factor IX and VIII deficiency that would be puzzling if the primary route of coagulation only flowed directly from fVII to fX.
1.3,2 The factor VII gene and cDNA
The cDNA for human factor VII was cloned in 1986 (Hagen et al. 1986). The full-length gene is comprised o f 9 exons spanning 12kb (Figure 1.8) and is located at chromosome 13q34, 2.8kb upstream of the factor X gene. It has a 1389bp coding region and a 5’ and 3 ’ non-coding region which are greater than 35bp and 1026bp respectively. Exon lb (66bp encoding amino acids -39 to -18) is absent from one of the cDNA clones characterised (Hagen et al. 1986). It is frequently absent from factor VII mRNA transcripts and over 90% of factor VII mRNA does not contain it (Berkner et al 1986).
Figure 1.8: General structure of the gene for human factor VII.
8
R1 R 2 R3 R4 R5
_ _ _ _ _ _ _ _ I_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I- - - 1- - - -
2000 4000 6000 8000 10000 12000
The locations o f the restriction sites used in sub-cloning the gene are shown (all o f the sites for the included restriction enzymes are shown). The positions of the eight
essential and one optional (exon lb) exons are shown as solid boxes [la= exon la -100 base pairs (bp) in length; lb = exon lb, 66bp; 2 = exon 2, 161bp; 3 = exon 3, 25 bp; 4 = exon 4, 139bp; 5 = exon 5, 141 bp; 6 = exon 6, 110 bp; 7 = exon 7, 124 bp; 8 = exon 8, 1600 bp] connected by lines representing the introns (A l, A2, B-G). The locations of five regions of minisatellite tandem repeats are shown as open boxes (R1-R5).
Reproduced from O ’Hara et al. (1987).
Full length cDNAs from rabbit (Brothers et al, 1993) and mouse (Idusogie et a l, 1996) and partial cDNAs from the rhesus monkey and dog have been characterised (Murakawa et a l, 1994). The synthesis of active factor VII is dependent on vitamin K and therefore, acquired factor VII deficiency is very common as levels of vitamin K fall rapidly during liver disease or upon commencement of oral anticoagulation with vitamin K antagonists. However, inherited factor VII deficiency is a rare, autosomal recessive disorder. The clinical features are quite variable with a rather poor correlation between the reported coagulant activity and clinical bleeding tendency (Ragni et a l, 1981, Triplett et a l, 1985). Thirty different point mutations and four short deletions have been described that cause fVII deficiency (Cooper et a l, 1997). O f the 30 point mutations, 26 are missense, 1 is nonsense (Figure 1.5) and 3 occur in donor splice sites o f introns 3, 4 and 7 (review by Cooper et al, 1997). Elevated levels o f factor VII have been associated with an increased risk of coronary thrombosis (Meade, 1983) and, paradoxically, there is also a report of several families in whom a decreased level of factor VII was also apparently associated with a thrombotic tendency (for a review see Goodnough fl/., 1983.)